Carbohydrate moieties attached to proteins and lipids in naturally-occurring glycoproteins and glycolipids play prominent roles in a number of biological activities such as cell adhesion, microbial infection, fertilization, cancer metastasis and intracellular transport. It is therefore useful to study these carbohydrate moieties by preparing oligosaccharide derivatives by conjugation for example with chromophores, fluorophores or radioactive isotopes at the reducing end of the sugar.
Glycoconjugate probes derived by chemical modification of oligosaccharides are powerful tools not only for the structural analysis and elucidation of biological function, but may also be useful in the development of conjugate vaccines, clinical diagnostics and therapeutics. Applications for chromophoric and fluorescent glycoconjugates include carbohydrate analysis, for example by HPLC, capillary electrophoresis and resolution on polyacrylamide gels, and as substrates for studies employing glycosyl transferase and glycoprotein-processing enzymes. Neoglycolipids are useful in probing of TLC plates with purified receptors known to recognize oligosaccharides and as glycolipid analogs (GLAs) as immunomodulators. Chemiluminescent derivatives are useful in chemiluminescent-based immunoassays. Glycoconjugate probes are also useful as immobilized sugars in solid-phase systems, as derivatives of biotin for formation of stable multivalent complexes with avidin, as neoglycopeptides and neoglycoproteins, as specific binding molecules in diagnostic reagents and as polysaccharide-conjugate vaccines.
The numerous applications for glycoconjugate probes have inspired significant research in chemical reactions and coupling methods for the modification of oligosaccharides. There are a number of known methods for the synthesis of glycoconjugates (Lee, Y. C. et al Glycoconjugates: Composition Structure and Function H. J. Allen & E. C. Kisaulis, eds; Dekker, New York; 121;1992).
For example, reductive amination is a widely used technique for the preparation of a glycoconjugate wherein the reducing terminus of the oligosaccharide is reacted with an amino group of protein to form a Schiff adduct. The Schiff adduct is subsequently reduced with sodium cyanoborohydride to form a covalent bond between the aldehyde form of the sugar and a primary amine. Reductive amination has been used for the synthesis of neoglycoproteins (Gray, G. R. Arch Biochem Biophys 163:426;1974) and fluorescent conjugates (Hase, S. et al J Biochem 85:995; 1979).
However, reductive amination results in degradation of the reducing end monosaccharide due to ring-opening of the pyranose residue which may adversely affect the biological activity or immunogenicity of the carbohydrate moieties for in vivo studies (Kamicker, B. S. et al Arch Biochem Biophys 183:393;1977). The reaction time is very slow, typically many days, even when the sugar is present in large excess, which excess is uneconomical and requires a significant sample clean-up procedure. Moreover, the resultant secondary ammonium linkage and the acyclic ring of these conjugates may exert undesirable influence resulting in questionable use of this method to prepare immunogens containing oligosaccharide haptens where highly specific anti-carbohydrate antibodies are desired (Danielson, S. J. et al Glycoconjugate J 3:363;1986). It will be appreciated by those skilled in the art that it is important for biological studies that the integrity of the glycan residue suffers minimum perturbation during any modification that leads to the preparation of a glycoconjugate.
Another method for synthesis is by direct fluorescent labelling with 2-aminopyridine wherein the reducing sugar is fluorescently labelled by condensing directly with 2-aminopyridine under strong acidic conditions (Her, G. R. et al J Carb Chem 6:129;1987). However, the yield is low, the reaction is slow and requires acidic conditions, excess reagents and high temperatures. These conditions encourage the formation of by-products due to the degradation of carbohydrates by the browning reaction.
Risley, J. M. et al (WO88/04323, Jun. 16, 1988) describes a method for direct derivatization of the 1-amino function of glycosylamines. 1-Amino-1-deoxyoligosaccharide is prepared from a glycopeptide or a glycoprotein using a .beta.-aspartylglycosylamineamidohydrolase. The resultant 1-amino-1-deoxyoligosaccharide is then reacted with a reactive acyl derivative, such as acid chloride or acid anhydride, at alkaline or neutral pH. The reaction occurs at the 1-amino functional group. However, acid chlorides are a very reactive species and there is a likelihood that O-acylation of the sugar hydroxyl groups will occur.
Manger, I. D. et al (Canadian Patent Application No. 2,023,339; published Feb. 17, 1991 and Canadian Patent Application Number 2,080,502; published Apr. 16, 1993) disclose a method wherein oligosaccharides are derivatized to form synthetic N-linked glycoconjugates by converting a glycosylamine derivative of the oligosaccharide to a haloacetylated derivative as an intermediate compound. Subsequent ammonolysis of the chloroacetamido function produces the corresponding 1-N-glycyl-.beta.-derivative which is used to synthesize N-linked glycoconjugates. The haloacetylation reaction is carried out by reaction of the glycosylamine with an excess, typically 5- to 10-fold, of chloroacetic anhydride.
The above-mentioned methods preserve the pyranose form of the reducing end sugar of oligosaccharides, unlike the reductive amination method which degrades the reducing end sugar. However, both methods involve direct functionalization of the 1-amino function, a process which is inefficient and difficult to control because of the poor nucleophilicity of the 1-amino group and the tendency of glycosylamines towards dimerization and hydrolysis. A comparison of the basicity of the 1-amino (pK.sub.a =5.2) and 2-amino (pK.sub.a =7.7) functions is suggestive of the poor utility of the 1-amino function as a nucleophile. Even though small electrophiles, such as acetic anhydride and chloroacetic anhydride, react in a better fashion, the use of excess reagents is necessary, for example a 10-fold excess of the N-acetylation reagent, in the method of Manger et al.
The main drawbacks of the prior art methods described above are that they require the use of either excess reagents or a multi-step procedure. These are significant drawbacks when dealing with minute amounts of biologically important oligosaccharides from natural glycoconjugates.
In view of the many applications for glycoconjugate derivatives, it is an object of the present invention to provide a method for producing oligosaccharide derivatives which overcomes the disadvantages of the prior art.